Production of soy protein product using calcium chloride extraction (&#34;s702&#34;)

ABSTRACT

A soy protein product having a protein content of at least about 60 wt % (N×6.25) d.b., preferably a soy protein isolate having a protein content of at least about 90 wt % (N×6.25) d.b., is prepared from a soy protein source material by extraction of the soy protein source material with an aqueous calcium salt solution, preferably calcium chloride solution, to cause solubilization of soy protein from the protein source and to form an aqueous soy protein solution, separating the aqueous soy protein solution from residual soy protein source, concentrating the aqueous soy protein solution while maintaining the ionic strength substantially constant by using a selective membrane technique, optionally diafiltering the concentrated soy protein solution, and drying the concentrated and optionally diafiltered soy protein solution.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC 119(e) from U.S.Provisional Patent Applications Nos. 61/202,262 filed Feb. 11, 2009 and61/213,663 filed Jun. 30, 2009.

FIELD OF INVENTION

The present invention is concerned with the preparation of soybeanprotein products.

BACKGROUND TO THE INVENTION

In U.S. Provisional Patent Applications Nos. 61/107,112 filed Oct. 21,2008, 61/193,457 filed Dec. 2, 2008, 61/202,070 filed Jan. 26, 2009,61/202,553 filed Mar. 12, 2009, 61/213,717 filed Jul. 7, 2009,61/272,241 filed Sep. 3, 2009, and U.S. patent application Ser. No.12/603,087 filed Oct. 21, 2009 the disclosures of which are incorporatedherein by reference, there is described the preparation of a soy proteinproduct, preferably a soy protein isolate, which is completely solubleand is capable of providing transparent and heat stable solutions at lowpH values. This soy protein product may be used for proteinfortification of, in particular, soft drinks and sports drinks, as wellas other acidic aqueous systems, without precipitation of protein. Thesoy protein product is produced by extracting a soy protein source withaqueous calcium chloride solution at natural pH, optionally diluting theresulting aqueous soy protein solution, adjusting the pH of the aqueoussoy protein solution to a pH of about 1.5 to about 4.4, preferably about2.0 to about 4.0, to produce an acidified clear soy protein solution,which may be optionally concentrated and/or diafiltered before drying.

SUMMARY OF THE INVENTION

It has now been found that calcium chloride extracts of soy proteinsource may be processed by alternative procedures to providesubstantially equivalent soy protein products, having a protein contentof at least about 60 wt % (N×6.25) d.b., that are soluble in acidicmedia and produce transparent, heat stable solutions at low pH values,and, therefore may be used for protein fortification of, in particular,soft drinks and sports drinks, as well as other aqueous systems, withoutprecipitation of protein. The soy protein product is preferably anisolate having a protein content of at least about 90 wt %, preferablyat least about 100% (N×6.25) d.b.

In one aspect of the present invention, a soy protein source material isextracted with aqueous calcium chloride solution at natural pH and theresulting aqueous soy protein solution is subjected to ultrafiltrationand optional diafiltration to provide a concentrated and optionallydiafiltered soy protein solution, which may be dried to provide the soyprotein product. The level of anti-nutritional trypsin inhibitors in thesoy protein product may be controlled by choosing the membraneprocessing conditions so as to release the desired amount of inhibitorsin the permeate stream.

In another aspect of the present invention, a soy protein sourcematerial is extracted with aqueous calcium chloride solution at naturalpH and the resulting aqueous soy protein solution is subjected toultrafiltration and optional diafiltration to provide a concentrated andoptionally diafiltered soy protein solution. This soy protein may befractionated by dilution into water, yielding a precipitate rich inglobulin proteins and a supernatant rich in albumin proteins. Thesupernatant may be processed, as described in detail below, to form soyprotein products having a soy protein content of at least about 60 wt %,preferably a soy protein isolate having a protein content of at leastabout 90 wt %. Trypsin inhibitors, which are proteins, are foundprimarily in the supernatant fraction after dilution. The precipitatefraction may be further processed or dried as is to provide the soyprotein product, but with a reduced level of trypsin inhibitors.

The soy protein isolate provided herein is soluble at acid pH values toprovide transparent and heat stable aqueous solutions thereof. The soyprotein isolate may be used for protein fortification of, in particular,soft drinks and sports drinks, without precipitation of protein.

In another aspect of the present invention, the concentrated andoptionally diafiltered soy protein solution, prepared as described aboveis diluted into water, but all the proteins are resolubilized byadjustment of the pH to about 1.5 to about 4.4, preferably about 2.0 toabout 4.0. The diluted and acidified solution may then be optionallyconcentrated and/or diafiltered. Reduction in the trypsin inhibitorlevel may be achieved by judicious choice of the membrane processingparameters or optionally employing a heat treatment step on theacidified solution.

In accordance with one aspect of the present invention, there isprovided a method of producing a soy protein product having a soyprotein content of at least 60 wt % (N×6.25), on a dry weight basis,which comprises:

-   -   (a) extracting a soy protein source with an aqueous calcium        chloride solution to cause solubilization of soy protein from        the protein source and to form an aqueous soy protein solution,    -   (b) separating the aqueous soy protein solution from residual        soy protein source,    -   (c) concentrating the aqueous soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (d) optionally diafiltering the concentrated soy protein        solution, and    -   (e) drying the concentrated soy protein solution.

The soy protein product is preferably an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

A variation of this procedure may be adopted to produce the product witha reduced content of albumin proteins and trypsin inhibitors. In such avariation, the concentrated and optionally diafiltered soy proteinsolution is diluted into water to yield a precipitate with a reducedcontent of albumin proteins and trypsin inhibitors. The precipitate maybe collected and dried to yield the product or the precipitate may besolubilized in water at low pH and then dried. Alternatively, thesolution formed by re-solubilizing the precipitate in water at low pHmay be optionally heat treated and/or concentrated and/or diafilteredbefore drying.

According to another aspect of the present invention, there is describeda method of producing a soy protein product having a soy protein contentof at least about 60 wt % (N×6.25), dry weight basis, which comprises:

-   -   (a) extracting a soy protein source with an aqueous calcium salt        solution to cause solubilization of soy protein from the protein        source and to form an aqueous soy protein solution,    -   (b) separating the aqueous soy protein solution from residual        soy protein source,    -   (c) concentrating the aqueous soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (d) optionally diafiltering the concentrated soy protein        solution,    -   (e) diluting the concentrated soy protein solution into water to        cause the formation of a precipitate,    -   (f) separating the precipitate from the diluting water, termed        the supernatant, and    -   (g) drying the separated soy protein precipitate.

Another variation of this procedure may be adopted to produce theproduct. In such a variation, the concentrated and optionallydiafiltered soy protein solution is diluted into water and the pHlowered. The resulting clear, acidified solution is optionallyconcentrated and/or diafiltered and/or heat treated before drying toyield the product.

According to a further aspect of the present invention, there isprovided a method of producing a soy protein product having a soyprotein content of at least about 60 wt % (N×6.25), dry weight basis,which comprises:

-   -   (a) extracting a soy protein source with an aqueous calcium salt        solution to cause solubilization of soy protein from the protein        source and to form an aqueous soy protein solution,    -   (b) separating the aqueous soy protein solution from residual        soy protein source,    -   (c) concentrating the aqueous soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (d) optionally diafiltering the concentrated soy protein        solution,    -   (e) diluting the concentrated soy protein solution into water to        cause the formation of a precipitate,    -   (f) acidifying the mixture of precipitate and diluting water to        re-solubilize the protein and form in a clear soy protein        solution,    -   (g) concentrating the clear acidified soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (h) optionally diafiltering the concentrated clear acidified soy        protein solution, and    -   (i) drying the concentrated and optionally diafiltered clear        acidified soy protein solution.

Employing the procedures of the present invention allows the option ofproduction of the soy protein product in a natural pH form. Generationof the soy protein product without an acidification step allows easier,safer and more economical processing, since there is no need for acidsand their handling. In addition, this procedure permits the beverageformulator to acidify the protein and beverage with the acidifying agentof their choice, given the differing strengths and flavour profiles ofvarious acids.

While the present invention refers mainly to the production of soyprotein isolates, it is contemplated that soy protein products of lesserpurity may be provided having similar properties to the soy proteinisolate. Such lesser purity products may have a protein concentration ofat least about 60% by weight (N×6.25) d.b.

The novel soy protein products of the invention can be blended withpowdered drinks for the formation of aqueous soft drinks or sportsdrinks by dissolving the same in water. Such blend may be a powderedbeverage.

The soy protein products provided herein may be provided as an aqueoussolution thereof having a high degree of clarity at acid pH values andwhich is heat stable at these pH values.

In another aspect of the present invention, there is provided an aqueoussolution of the soy product provided herein which is heat stable at lowpH. The aqueous solution may be a beverage, which may be a clearbeverage in which the soy protein product is completely soluble andtransparent or an opaque beverage in which the soy protein product doesnot increase the opacity.

The soy protein products produced according to the processes herein lackthe characteristic beany flavour of soy protein isolates and aresuitable, not only for protein fortification of acid medium, but may beused in a wide variety of conventional applications of protein isolates,including, but not limited to protein fortification of processed foodsand beverages, emulsification of oils, as a body former in baked goodsand foaming agent in products which entrap gases. In addition, the soyprotein product may be formed into protein fibers, useful in meatanalogs and may be used as an egg white substitute or extender in foodproducts where egg white is used as a binder. The soy protein productmay be used as a nutritional supplement. Other uses of the soy proteinproduct are in pet foods, animal feed and in industrial and cosmeticapplications and in personal care products.

GENERAL DESCRIPTION OF INVENTION

The initial step of the process of providing the soy protein productinvolves solubilizing soy protein from a soy protein source. The soyprotein source may be soybeans or any soy product or by-product derivedfrom the processing of soybeans including but not limited to soy meal,soy flakes, soy grits and soy flour. The soy protein source may be usedin the full fat form, partially defatted form or fully defatted form.Where the soy protein source contains an appreciable amount of fat, anoil-removal step generally is required during the process. The soyprotein recovered from the soy protein source may be the proteinnaturally occurring in soybean or the proteinaceous material may be aprotein modified by genetic manipulation but possessing characteristichydrophobic and polar properties of the natural protein.

Protein solubilization from the soy protein source material is effectedmost conveniently using food grade calcium chloride solution, althoughsolutions of other calcium salts may be used. Where the soy proteinproduct is intended for non-food uses, non-food-grade chemicals may beused. In addition, other alkaline earth metal salts may be also used,such as magnesium salts. Further, extraction of the soy protein from thesoy protein source may also be effected using calcium salt solution incombination with another salt solution, such as sodium chloride.Additionally, extraction of the soy protein from the soy protein sourcemay be effected using water or other salt solution, such as sodiumchloride solution, with calcium salt, such as calcium chloride,subsequently being added to the aqueous soy protein solution produced inthe extraction step. Precipitate formed upon addition of the calciumsalt then is removed prior to subsequent processing.

As the concentration of the calcium salt solution increases, the degreeof solubilization of protein from the soy protein source initiallyincreases until a maximum value is achieved. Any subsequent increase insalt concentration does not increase the total protein solubilized. Theconcentration of the calcium salt solution which causes maximum proteinsolubilization varies depending on the salt concerned. It is usuallypreferred to utilize a concentration value less than about 1.0 M, andmore preferably a value of about 0.10 M to about 0.15 M.

In a batch process, the salt solubilization of the protein is effectedat a temperature of from about 1° C. to about 100° C., preferably about15° C. to about 35° C., preferably accompanied by agitation to decreasethe solubilization time, which is usually about 1 to about 60 minutes.It is preferred to effect the solubilization to extract substantially asmuch protein from the soy protein source as is practicable, so as toprovide an overall high product yield.

In a continuous process, the extraction of the soy protein from the soyprotein source is carried out in any manner consistent with effecting acontinuous extraction of soy protein from the soy protein source. In oneembodiment, the soy protein source is continuously mixed with thecalcium salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction in accordance with theparameters described herein. In such a continuous procedure, the saltsolubilization step is effected rapidly, in a time of up to about 10minutes, preferably to effect solubilization to extract substantially asmuch protein from the soy protein source as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 1° C. and about 100° C., preferably between about 15° C.and about 35° C.

The extraction is generally conducted at a p11 of about 5 to about 11,preferably about 5 to about 7. The pH of the extraction system (soyprotein source and calcium salt solution) may be adjusted, if necessary,to any desired value within the range of about 5 to about 11 for use inthe extraction step by the use of any convenient acid, usuallyhydrochloric acid, or alkali, usually sodium hydroxide, as required.

The concentration of soy protein source in the calcium salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 50 g/L, preferably about 10 toabout 50 L.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in the soyprotein source, which then results in the fats being present in theaqueous phase.

The aqueous calcium salt solution may contain an antioxidant. Theantioxidant may be any convenient antioxidant, such as sodium sulfite orascorbic acid. The quantity of antioxidant employed may vary from about0.01 to about 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit the oxidation of any phenolics in theprotein solution.

The aqueous phase resulting from the extraction step then may beseparated from the residual soy protein source, in any convenientmanner, such as by employing a decanter centrifuge, followed by disccentrifugation and/or filtration, to remove residual soy protein sourcematerial. The separated residual protein source material may be driedfor disposal. Alternatively, the separated residual soy protein sourcemay be processed to recover some residual protein, such as by aconventional isoelectric precipitation procedure or any other convenientprocedure to recover such residual protein.

Where the soy protein source contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein solution. Alternatively, defatting of theseparated aqueous protein solution may be achieved by any otherconvenient procedure.

The aqueous soy protein solution may be treated with an adsorbent, suchas powdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the separated aqueous protein solution. For powderedactivated carbon, an amount of about 0.025% to about 5% w/v, preferablyabout 0.05% to about 2% w/v, is employed. The adsorbing agent may beremoved from the soy solution by any convenient means, such as byfiltration.

If of adequate purity, the resulting aqueous soy protein solution may bedirectly dried to produce a soy protein product. To decrease theimpurities content, the aqueous soy protein solution may be processedprior to drying.

The aqueous soy protein solution may be concentrated to increase theprotein concentration thereof while maintaining the ionic strengththereof substantially constant. Such concentration generally is effectedto provide a concentrated soy protein solution having a proteinconcentration of about 50 to about 400 g/L, preferably about 100 toabout 250 g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the foodgrade salt but also low molecular weight materials extracted from thesource material, such as carbohydrates, pigments, low molecular weightproteins and anti-nutritional factors, such as trypsin inhibitors, whichare themselves low molecular weight proteins. The molecular weightcut-off of the membrane is usually chosen to ensure retention of asignificant proportion of the protein in the solution, while permittingcontaminants to pass through having regard to the different membranematerials and configurations.

The concentrated soy protein solution then may be subjected to adiafiltration step, before or after complete concentration, usingcalcium salt solution, such as a solution of calcium chloride at thesame pH and the same concentration of calcium salt as the extractionsolution. If a reduction in the salt content of the retentate isdesired, the diafiltration solution employed may be an aqueous calciumsalt solution at the same pH but lower salt concentration than theextraction solution. However, the salt concentration of thediafiltration solution must be chosen so that the salt level in theretentate remains sufficiently high to maintain the desired proteinsolubility. As mentioned, the diafiltration solution is preferably at apH equal to that of the protein solution being diafiltered. The pH ofthe diafiltration solution may be adjusted with any convenient acid,such as hydrochloric acid or phosphoric acid or alkali, such as sodiumhydroxide. Such diafiltration may be effected using from about 2 toabout 40 volumes of diafiltration solution, preferably about 5 to about25 volumes of diafiltration solution. In the diafiltration operation,further quantities of contaminants are removed from the aqueous soyprotein solution by passage through the membrane with the permeate. Thediafiltration operation may be effected until no significant furtherquantities of contaminants or visible colour are present in the permeateor until the retentate has been sufficiently purified so as, when dried,to provide a soy protein product with the desired protein content,preferably an isolate with a protein content of at least about 90 wt %on a dry weight basis. Such diafiltration may be effected using the samemembrane as for the concentration step. However, if desired, thediafiltration step may be effected using a separate membrane with adifferent molecular weight cut-off, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 1,000,000Daltons, preferably about 5,000 to about 100,000 Daltons, having regardto different membrane materials and configuration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6,25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity. The soyprotein product is still able to produce clear protein solutions underacidic conditions,

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %, The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 2° to about 60° C., preferablyabout 20° to about 35° C., and for the period of time to effect thedesired degree of concentration and diafiltration. The temperature andother conditions used to some degree depend upon the membrane equipmentused to effect the membrane processing, the desired proteinconcentration of the solution and the efficiency of the removal ofcontaminants to the permeate.

There are two main trypsin inhibitors in soy, namely the Kunitzinhibitor, which is a heat-labile molecule with a molecular weight ofapproximately 21,000 Daltons, and the Bowman-Birk inhibitor, a moreheat-stable molecule with a molecular weight of about 8,000 Daltons. Thelevel of trypsin inhibitor activity in the final soy protein product canbe controlled by manipulation of various process variables.

For example, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such asabout 30,000 to about 1,000,000 Daltons, operating the membrane atelevated temperatures, such as about 30° C. to about 60° C. andemploying greater volumes of diafiltration medium, such as about 20 toabout 40 volumes.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the soyprotein source material in the extraction step, may be added to theclarified aqueous soy protein solution following removal of residual soyprotein source material, may be added to the concentrated proteinsolution before or after diafiltration or may be dry blended with thedried soy protein product. The addition of the reducing agent may becombined with the membrane processing steps, as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by utilizing a concentration anddiafiltration membrane with a smaller pore size, operating the membraneat lower temperatures, employing fewer volumes of diafiltration mediumand not employing a reducing agent.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076. Alternatively, defatting of theconcentrated and optionally diafiltered protein solution may be achievedby any other convenient procedure.

The concentrated and optionally diafiltered aqueous protein solution maybe treated with an adsorbent, such as powdered activated carbon orgranulated activated carbon, to remove colour and/or odour compounds.Such adsorbent treatment may be carried out under any convenientconditions, generally at the ambient temperature of the concentratedaqueous protein solution. For powdered activated carbon, an amount ofabout 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, isemployed. The adsorbent may be removed from the soy protein solution byany convenient means, such as by filtration.

The concentrated and optionally diafiltered soy protein solutionresulting from the optional defatting and optional adsorbent treatmentstep may be subjected to a pasteurization step to reduce the microbialload. Such pasteurization may be effected under any desiredpasteurization conditions. Generally, the concentrated and optionallydiafiltered soy protein solution is heated to a temperature of about 55°to about 70° C., preferably about 60 to about 65′C, for about 30 secondsto about 60 minutes, preferably about 10 to about 15 minutes. Thepasteurized concentrated soy protein solution then may be cooled fordrying or further processing, preferably to a temperature of about 15°to about 35° C.

In accordance with one aspect of the current invention, the concentratedand optionally diafiltered clear aqueous soy protein solution may bedried by any convenient technique, such as spray drying or freeze dryingto yield the soy protein product. Alternatively, the concentrated andoptionally diafiltered soy protein solution may be adjusted in pH toabout 2.0 to about 4.0. The pH adjustment may be effected in anyconvenient manner, such as by the addition of hydrochloric acid orphosphoric acid. The resulting acidified soy protein solution then isdried. As a further alternative, the pH adjusted soy protein solutionmay be subjected to a heat treatment to inactivate heat labileanti-nutritional factors, such as the trypsin inhibitors mentionedabove. Such a heating step also provides the additional benefit ofreducing the microbial load. Generally, the protein solution is heatedto a temperature of about 70° to about 120° C., preferably about 85° toabout 95° C., for about 10 seconds to about 60 minutes, preferably about30 seconds to about 5 minutes. The heat treated acidified soy proteinsolution then may be cooled to a temperature of about 2° C. to about 60°C., preferably about 20° to about 35° C. The resulting acidified, heattreated soy protein solution then is dried.

In another aspect of the invention, the concentrated protein solutionresulting from the concentration step and optional diafiltration step,optional defatting step, optional adsorbent treatment step and optionalpasteurization step, is diluted to effect precipitate formation bymixing the concentrated protein solution with water having the volumerequired to achieve the degree of dilution desired. When theprecipitated protein is to be separated from the residual aqueous phase,termed the supernatant, as is the case for this aspect of the currentinvention, the degree of dilution is generally about 5 fold to about 25fold, preferably about 10 fold to about 20 fold. The water with whichthe concentrated protein solution is mixed preferably has a temperatureof about 1° to about 60° C., preferably about 15° to about 35° C.

In a batch operation, the batch of concentrated protein solution isadded to a static body of water having the desired volume, as discussedabove. The dilution of the concentrated protein solution andconsequential decrease in ionic strength causes the formation of theprotein precipitate. In the batch procedure, the protein precipitate isallowed to settle in the body of water. The settling may be assisted,such as by centrifugation. Such induced settling decreases the moisturecontent and the occluded salt content of the precipitated protein.

Alternatively, the dilution operation may be carried out continuously bycontinuously passing the concentrated protein solution to one inlet of aT-shaped pipe, while the diluting water is fed to the other inlet of theT-shaped pipe, permitting mixing in the pipe. The diluting water is fedinto the T-shaped pipe at a rate sufficient to achieve the desireddegree of dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and the diluting waterin the pipe initiates the formation of protein precipitate and themixture is continuously fed from the outlet of the T-shaped pipe into asettling vessel, from which, when full, supernatant is permitted tooverflow. The mixture preferably is fed into the body of liquid in thesettling vessel in a manner which minimizes turbulence within the bodyof liquid.

In the continuous procedure, the protein precipitate is allowed tosettle in the settling vessel and the procedure is continued until adesired quantity of the precipitate has accumulated in the bottom of thesettling vessel, whereupon the accumulated precipitate is removed fromthe settling vessel. In lieu of settling by sedimentation, theprecipitate may be separated continuously by centrifugation.

By the utilization of a continuous process for the recovery of soyprotein precipitate as compared to the batch process, the initialprotein extraction step can be significantly reduced in time for thesame level of protein extraction. In addition, in a continuousoperation, there is less chance of contamination than in a batchprocedure, leading to higher product quality and the process can becarried out in more compact equipment.

The settled precipitate is separated from the residual aqueous phase orsupernatant, such as by decantation of the residual aqueous phase fromthe settled mass or by centrifugation. The precipitate may be washed toremove residual supernatant, such as with about 1 to about 10,preferably about 2 to about 3 volumes of water and then the precipitaterecovered again, as above. The optionally washed precipitate may be usedin the wet form or may be dried, by any convenient technique, such asspray drying or freeze drying, to a dry form. The dry precipitate has ahigh protein content, in excess of about 60 wt % protein, preferably atleast about 90 wt % protein (N×6.25), and more preferably at least about100 wt % (N×6.25). The dry precipitate is low in phytic acid content,generally less than about 1.5% by weight.

The supernatant arising from the dilution step may be discarded or, ifof sufficient purity, dried to produce a soy protein product. Todecrease the impurities content, the supernatant may be processed, withacidification, such as to pH about 1.5 to about 4.4, preferably about2.0 to about 4.0, or without acidification and dried by any convenientmeans to yield one or more soy protein products. The supernatant streamis enriched in trypsin inhibitors due to the fractionation occurring ondilution. The supernatant may be processed to yield a dry proteinproduct high in trypsin inhibitor activity or the process steps may begeared to reduce the trypsin inhibitor activity of the protein derivedfrom this stream. If processed without acidification, heat treatment ofthe supernatant before or after concentration may be employed toprecipitate a fraction of heat sensitive proteins, while the trypsininhibitors stay largely in solution. Alternatively, the supernatant maybe concentrated at low pH and then the sample adjusted in pH to about 6to about 7, using any convenient alkali, such as sodium hydroxide, priorto the application of the heat treatment to precipitate the heatsensitive proteins. Such a heat treatment may be effected at atemperature of about 70° C. to about 120° C., preferably about 75° C. toabout 105° C. for about 1 minute to about 30 minutes, preferably about 5minutes to about 15 minutes. The heat precipitated proteins may beremoved in any convenient manner, such as centrifugation or filtrationor a combination thereof. The precipitate then may be washed with about1 to about 10, preferably about 2 volumes of water to remove entrappedsupernatant, then recovered as above and dried by any convenient meansto provide a soy protein product with a reduced trypsin inhibitorcontent.

Heat treatment of the acidified supernatant may be used to inactivateheat-labile trypsin inhibitors. Partially concentrated or fullyconcentrated acidified soy protein solution may also be heat treated toinactivate heat labile trypsin inhibitors. Generally, the proteinsolution is heated to a temperature of about 70° to about 120° C.,preferably about 85° to about 95° C., for about 10 seconds to about 60minutes, preferably about 30 seconds to about 5 minutes. The heattreated acidified soy protein solution then may be cooled to atemperature of about 2° C. to about 60° C., preferably about 20° toabout 35° C. for further processing.

The supernatant or the acidified and optionally heat treated supernatantor the centrate resulting from the removal of proteins deposited by heattreatment of the supernatant, which may optionally be acidified afterthe removal of the precipitated protein, such as to pH about 1.5 toabout 4.4, preferably about 2.0 to about 4.0, may be concentrated toincrease the protein concentration thereof. Such concentration iseffected using any convenient selective membrane technique, such asultrafiltration or diafiltration, using membranes with a suitablemolecular weight cut-off permitting low molecular weight species,including salt, carbohydrates, pigments, trypsin inhibitors and otherlow molecular weight materials extracted from the protein sourcematerial, to pass through the membrane, while retaining a significantproportion of the soy protein in the solution. Ultrafiltration membraneshaving a molecular weight cut-off of about 3,000 to 1,000,000 Daltons,preferably about 5,000 to about 100,000 Daltons, having regard todiffering membrane materials and configuration, may be used.Concentration of the protein solution in this way also reduces thevolume of liquid required to be dried to recover the protein. Theprotein solution generally is concentrated to a protein concentration ofabout 50 g/L to about 400 g/L, preferably about 100 to about 250 g/L,prior to drying. Such concentration operation may be carried out in abatch mode or in a continuous operation, as described above.

The soy protein solution may be subjected to a diafiltration step,before or after complete concentration, using water or a dilute saltsolution. The water or dilute salt solution may be at its natural pH orat a pH equal to that of the protein solution being diafiltered or atany pH value in between. Such diafiltration may be effected using fromabout 2 to about 40 volumes of diafiltration solution, preferably about5 to about 25 volumes of diafiltration solution. In the diafiltrationoperation, further quantities of contaminants are removed from the clearaqueous soy protein solution by passage through the membrane with thepermeate. The diafiltration operation may be effected until nosignificant further quantities of contaminants or visible colour arepresent in the permeate or until the protein solution has beensufficiently purified so as, when dried, to provide a soy proteinproduct with the desired protein content, preferably an isolate with aprotein content of at least 90 wt % (N×6.25) d.b. Such diafiltration maybe effected using the same membrane as for the concentration step.However, if desired, the diafiltration step may be effected using aseparate membrane with a different molecular weight cutoff, such as amembrane having a molecular weight cut-off in the range of about 3,000to about 1,000,000 Daltons, preferably about 5,000 to about 100,000Daltons, having regard to different membrane materials andconfiguration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6.25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The concentration step and the optional diafiltration step may beeffected at any convenient temperature, generally about 2° C. to about60° C., preferably about 20° C. to about 35° C., and for the period oftime to effect the desired degree of concentration and diafiltration.The temperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

The concentration and/or diafiltration steps may be operated in a mannerfavorable for removal of trypsin inhibitors in the permeate along withthe other contaminants. Removal of the trypsin inhibitors is promoted byusing a membrane of larger pore size, such as 30,000 to 1,000,000Daltons, operating the membrane at elevated temperatures, such as 30 to60° C. and employing greater volumes of diafiltration medium, such as 20to 40 volumes.

Acidifying and membrane processing the protein solution at a lower pH(1.5 to 3) may also reduce the trypsin inhibitor activity relative toprocessing the solution at a higher pH (3 to 4.4) or withoutacidification. When the protein solution is concentrated and diafilteredat the low end of the pH range, it may be desired to raise the pH of theretentate prior to drying. The pH of the concentrated and diafilteredprotein solution may be raised to the desired value, for example pH 3,by the addition of any convenient food grade alkali such as sodiumhydroxide.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added to thesupernatant or the centrate arising from a heat precipitation step, maybe added to the concentrated solution before or after diafiltration ormay be dry blended with the dried soy protein product. The addition ofthe reducing agent may be combined with a heat treatment step and themembrane processing steps, as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by eliminating or reducing theintensity of the heat treatment step, not utilizing reducing agents,operating the concentration and diafiltration steps at higher pH values,utilizing a concentration and diafiltration membrane with a smaller poresize, operating the membrane at lower temperatures and employing fewervolumes of diafiltration medium.

The concentrated and optionally diafiltered aqueous protein solution maybe treated with an adsorbent, such as powdered activated carbon orgranulated activated carbon, to remove colour and/or odour compounds.Such adsorbent treatment may be carried out under any convenientconditions, generally at the ambient temperature of the proteinsolution. For powdered activated carbon, an amount of about 0.025% toabout 5% w/v, preferably about 0.05% to about 2% w/v, is employed. Theadsorbent may be removed from the soy protein solution by any convenientmeans, such as by filtration.

The concentrated and optionally diafiltered aqueous soy protein solutionthen may be dried by any convenient technique, such as spray drying orfreeze drying. The dry soy protein product has a protein content of atleast about 60 wt % (N×6.25) d.b., preferably in excess of about 90 wt %(N×6.25) d,b., more preferably at least about 100 wt % (N×6.25), d.b.The soy protein product is low in phylic acid content, generally lessthan about 1.5% by weight.

As mentioned above, the settled protein precipitate formed in thedilution step may be directly dried to yield the protein product.Alternatively, the wet protein precipitate may be re-suspended in water,such as about 2 to about 3 volumes, and re-solubilized by adjusting thepH of the sample to about 1.5 to about 4.4, preferably about 2.0 toabout 4.0, using any convenient acid, such as hydrochloric acid orphosphoric acid. The clear protein solution then may be dried by anyconvenient technique, such as spray drying or freeze drying to a dryform. The dry protein product has a protein content in excess of about60 wt % protein, preferably at least about 90 wt % protein, morepreferably at least about 100 wt % protein (N×6.25)

As a further alternative, the clear, acidified, re-solubilized soyprotein solution may be subjected to a heat treatment to inactivate anyremaining heat labile anti-nutritional factors. Such a heating step alsoprovides the additional benefit of reducing the microbial load.Generally, the protein solution is heated to a temperature of about 70°to about 120° C., preferably about 85′ to about 95° C., for about 10seconds to about 60 minutes, preferably about 30 seconds to about 5minutes. The heat treated, acidified soy protein solution then may becooled for further processing as described below, to a temperature ofabout 2° to about 60° C., preferably about 20° to about 35° C.

The acidified and optionally heat treated clear solution, may beconcentrated to increase the protein concentration thereof. Suchconcentration is effected using any convenient selective membranetechnique, such as ultrafiltration or diafiltration, using membraneswith a suitable molecular weight cut-off permitting low molecular weightspecies, including salt, carbohydrates, pigments, trypsin inhibitors andother low molecular weight materials extracted from the protein sourcematerial, to pass through the membrane, while retaining a significantproportion of the soy protein in the solution. Ultrafiltration membraneshaving a molecular weight cut-off of about 3,000 to 1,000,000 Daltons,preferably about 5,000 to about 100,000 Daltons, having regard todiffering membrane materials and configuration, may be used.Concentration of the protein solution in this way also reduces thevolume of liquid required to be dried to recover the protein. Theprotein solution generally is concentrated to a protein concentration ofabout 50 g/L to about 300 g/L, preferably about 100 to about 200 g/L,prior to drying. Such concentration operation may be carried out in abatch mode or in a continuous operation, as described above.

The soy protein solution may be subjected to a diafiltration step beforeor after complete concentration using water. The water may be at itsnatural pH or at a pH equal to that of the protein solution beingdiafiltered or at any pH value in between. Such diafiltration may beeffected using from about 2 to about 40 volumes of diafiltrationsolution, preferably about 5 to about 25 volumes of diafiltrationsolution. In the diafiltration operation, further quantities ofcontaminants are removed from the clear aqueous soy protein solution bypassage through the membrane with the permeate. The diafiltrationoperation may be effected until no significant further quantities ofcontaminants or visible colour are present in the permeate or until theretentate has been sufficiently purified so as, when dried, to provide asoy protein product with the desired protein content, preferably anisolate with a protein content of at least about 90 wt % (N×6.25) d.b.Such diafiltration may be effected using the same membrane as for theconcentration step. However, if desired, the diafiltration step may beeffected using a separate membrane with a different molecular weightcut-off, such as a membrane having a molecular weight cut-off in therange of about 3,000 to about 1,000,000 Daltons, preferably about 5,000to about 100,000 Daltons, having regard to different membrane materialsand configuration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6.25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity. The soyprotein product is still able to produce clear protein solutions underacidic conditions.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The optional concentration step and the optional diafiltration step maybe effected at any convenient temperature, generally about 2° to about60° C., preferably about 20° to about 35° C., and for the period of timeto effect the desired degree of concentration and diafiltration. Thetemperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

As mentioned above, the level of trypsin inhibitor activity in the finalsoy protein product can be controlled by manipulation of various processvariables,

As previously noted, heat treatment of the acidified aqueous soy proteinsolution may be used to inactivate heat-labile trypsin inhibitors.Partially concentrated or fully concentrated acidified soy proteinsolution may also be heat treated to inactivate heat labile trypsininhibitors.

In addition, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such as30,000 to 1,000,000 Daltons, operating the membrane at elevatedtemperatures, such as 30° to 60° C. and employing greater volumes ofdiafiltration medium, such as 20 to 40 volumes.

Acidifying and membrane processing the protein solution at a lower pH(1.5 to 3) may reduce the trypsin inhibitor activity relative toprocessing the solution at higher pH (3 to 4.4). When the proteinsolution is concentrated and diafiltered at the low end of the pH range,it may be desired to raise the pH of the retentate prior to drying. ThepH of the concentrated and diafiltered protein solution may be raised tothe desired value, for example pH 3, by the addition of any convenientfood grade alkali such as sodium hydroxide.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added to the wetprotein precipitate resulting from the dilution step, may be added tothe protein solution formed by acidifying and re-solubilizing theprecipitate, may be added to the concentrated solution before or afterdiafiltration or may be dry blended with the dried soy protein product.The addition of the reducing agent may be combined with a heat treatmentstep and the membrane processing steps, as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by eliminating or reducing theintensity of the heat treatment step, not utilizing reducing agents,operating the concentration and diafiltration steps at the higher end ofthe pH range (3 to 4.4), utilizing a concentration and diafiltrationmembrane with a smaller pore size, operating the membrane at lowertemperatures and employing fewer volumes of diafiltration medium.

The acidified, optionally concentrated and optionally diafiltered clearaqueous protein solution may be treated with an adsorbent, such aspowdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the protein solution. For powdered activated carbon, anamount of about 0.025% to about 5% w/v, preferably about 0.05% to about2% is employed. The adsorbent may be removed from the soy proteinsolution by any convenient means, such as by filtration.

The acidified, optionally concentrated and optionally diafiltered clearaqueous soy protein solution then may be dried by any convenienttechnique, such as spray drying or freeze drying. The dry soy proteinproduct has a protein content of at least about 60 wt % (N×6.25) d.b.,preferably in excess of about 90 wt % (N×6.25) d.b., more preferably atleast about 100 wt % (N×6.25) d.b. The soy protein product is low inphytic acid content, generally less than about 1.5% by weight.

In accordance with another aspect of the current invention, the proteinprecipitated upon dilution into water may be processed together with thesupernatant. In such a case, the degree of dilution is generally about 1to 25 fold, preferably about 3 to about 12 fold. The water with whichthe concentrated protein solution is mixed has a temperature of about 1°to about 60° C., preferably about 15° C. to about 35° C.

The dilution water, containing the deposited protein precipitate, isadjusted in pH to about 1.5 to about 4.4, preferably about 2.0 to about4.0, using any convenient acid, such as hydrochloric acid or phosphoricacid. The drop in pH causes the resolubilization of the proteindeposited by dilution yielding a clear, acidified protein solution. Theprotein solution may be used in the wet form or may be dried, by anyconvenient technique, such as spray drying or freeze drying, to a dryform.

As a further alternative, the protein solution formed by acidifying themixture of protein precipitate and supernatant may be processedutilizing the same steps as described above for the isolated precipitateresolubilized by acidification.

The optionally concentrated, optionally diafiltered, optionally heattreated, optional adsorbent treated clear aqueous soy protein solutionthen may be dried by any convenient technique, such as spray drying orfreeze drying. The dry soy protein product has a protein content inexcess of about 60 wt % protein, preferably at least about 90 wt %, morepreferably about 100 wt % (N×6.25) d.b.

The soy protein products produced herein are soluble in an acidicaqueous environment, making the products ideal for incorporation intobeverages, both carbonated and uncarbonated, to provide proteinfortification thereto. Such beverages have a wide range of acidic pHvalues, ranging from about 2.5 to about 5. The soy protein productsprovided herein may be added to such beverages in any convenientquantity to provide protein fortification to such beverages, forexample, at least about 5 g of the soy protein per serving. The addedsoy protein product dissolves in the beverage and does not impair theclarity of the beverage, even after thermal processing.

The soy protein product may be blended with dried beverage prior toreconstitution of the beverage by dissolution in water. In some cases,modification of the normal formulation of the beverage to tolerate thecomposition of the invention may be necessary where components presentin the beverage may adversely affect the ability of the composition toremain dissolved in the beverage.

EXAMPLES Example 1

This Example illustrates the production of a soy protein isolate that issoluble, transparent and heat stable in acidic solutions and is membraneprocessed at natural pH. The production of this isolate does not involvea dilution step.

20 kg of defatted, minimally heat processed soy flour was added to 200 Lof 0.15 M CaCl₂ solution at ambient temperature and agitated for 30minutes to provide an aqueous protein solution. The residual soy flourwas removed and the resulting protein solution was clarified bycentrifugation and filtration to produce 169 L of filtered proteinsolution having a protein content of 1.68% by weight.

The filtered protein extract solution was reduced in volume to 31 L byconcentration on a PVDF membrane having a molecular weight cutoff of5,000 Daltons. The concentrated protein solution was diafiltered with 62L of 0.075M CaCl₂. The resulting diafiltered, concentrated proteinsolution had a protein content of 13.28% by weight and represented ayield of 95.2 wt % of the initial filtered protein solution. Thediafiltered, concentrated protein solution was then dried to yield aproduct found to have a protein content of 91.45% (N×6.25) d.b. Theproduct was termed S005-L11-08A S702.

A 3.2% w/v protein solution of 5702 was prepared in water and the pHlowered to 3 with diluted HCl. The colour and clarity was then assessedusing a HunterLab ColorQuest XE instrument operated in transmissionmode.

The colour and clarity values are set forth in the following Table 1:

TABLE 1 HunterLab scores for 3.2% protein solution of S005-L11-08A S702at pH 3 sample L* a* b* haze (%) S702 96.51 −0.82 11.45 0.8

As may be seen from Table 1, the colour of the S702 solution at pH 3 wasvery light and the haze level was very low.

The colour of the dry powder was also assessed with the HunterLabColorQuest XE instrument in reflectance mode. The colour values are setforth in the following Table 2:

TABLE 2 HunterLab scores for S005-L11-08A S702 dry powder sample L* a*b* S702 85.11 0.37 11.11

As may be seen from Table 2, the dry colour of the S702 powder was verylight.

The trypsin inhibitor activity of the isolate was determined using themethod of Kakade et al, Cereal Chem., 51:376-381 (1974), TheS005-L11-08A S702 was found to have a trypsin inhibitor activity of 87trypsin inhibitor units (TIU)/mg protein (N×6.25).

Example 2

This Example contains an evaluation of the heat stability in water ofthe soy protein isolate produced by the method of Example 1 (S702).

A 2% w/v protein solution of S005-L11-08A S702 in water was produced andthe pH adjusted to 3. The clarity of this solution was assessed by hazemeasurement with the HunterLab ColorQuest XE instrument. The solutionwas then heated to 95° C., held at this temperature for 30 seconds andthen immediately cooled to room temperature in an ice bath. The clarityof the heat treated solution was then measured again.

The clarity of the protein solution before and after heating is setforth in the following Table 3:

TABLE 3 Effect of heat treatment on clarity of S702 solution sample haze(%) before heating 5.0 after heating 0.6

As can be seen from the data in Table 3, the sample was heat stable. Theprotein solution was initially very clear and the heat treatmentactually reduced the level of haze.

Example 3

This Example contains an evaluation of the solubility in water of thesoy protein isolate produced by the method of Example 1 (S702).Solubility was tested based on protein solubility (termed proteinmethod, a modified version of the procedure of Mon et al., J. Food Sci.50:17154718) and total product solubility (termed pellet method).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared atnatural pH. For the pH adjusted samples, the pH was measured andcorrected two times during the 60 minutes stirring. After the 60 minutesof stirring, the samples were made up to 50 ml total volume with ROwater, yielding a 1% w/v protein dispersion. The protein content of thedispersions was measured using a Leco FP528 Nitrogen Determinator.Aliquots (20 ml) of the dispersions were then transferred to pre-weighedcentrifuge tubes that had been dried overnight in a 100° C. oven thencooled in a desiccator and the tubes capped. The samples werecentrifuged at 7800 g for 10 minutes, which sedimented insolublematerial and yielded a clear supernatant. The protein content of thesupernatant was measured by Leco analysis and then the supernatant andthe tube lids were discarded and the pellet material dried overnight inan oven set at 100° C. The next morning the tubes were transferred to adesiccator and allowed to cool. The weight of dry pellet material wasrecorded. The dry weight of the initial protein powder was calculated bymultiplying the weight of powder used by a factor of ((100−moisturecontent of the powder (%))/100). Solubility of the product was thencalculated two different ways:

1) Solubility (protein method) (%) (% protein in supernatant/% proteinin initial dispersion)×100

2) Solubility (pellet method) (%) (1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100

The natural pH value of the protein isolate produced in Example 1 inwater (1% protein) is shown in Table 4:

TABLE 4 Natural pH of S702 solution prepared in water at 1% proteinBatch Product Natural pH S005-L11-08A S702 5.91

The solubility results obtained are set forth in the following Tables 5and 6:

TABLE 5 Solubility of S702 at different pH values based on proteinmethod Solubility (protein method) (%) Batch Product pH 2 pH 3 pH 4 pH 5pH 6 pH 7 Nat. pH S005- S702 98.2 95.8 100 94.2 15.1 11.2 10.9 L11-08A

TABLE 6 Solubility of S702 at different pH values based on pellet methodSolubility (pellet method) (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH7 Nat. pH S005- S702 98.5 100 97.3 26.6 10.2 32.0 28.3 L11-08A

As can be seen from the results of Tables 5 and 6, the S702 productswere very soluble in the pH range of 2 to 4.

Example 4

This Example contains an evaluation of the clarity in water of the soyprotein isolate produced by the method of Example 1 (S702).

The clarity of the 1% w/v protein solution prepared as described inExample 3 was assessed by measuring the absorbance at 600 nm, with alower absorbance score indicating greater clarity. Analysis of thesamples on a HunterLab ColorQuest XE instrument in transmission modealso provided a percentage haze reading, another measure of clarity.

101161 The clarity results are set forth in the following Tables 7 and8:

TABLE 7 Clarity of S702 solution at different pH values as assessed byA600 A600 Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH S005- S7020.012 0.019 0.094 >3.0 2.201 2.422 2.283 L11-08A

TABLE 8 Clarity of S702 solution at different pH values as assessed byHunterLab analysis HunterLab haze reading (%) Batch Product pH 2 pH 3 pH4 pH 5 pH 6 pH 7 Nat. pH S005- S702 0.0 2.2 16.0 97.3 97.6 100.1 101.9L11-08A

As can be seen from the results of Tables 8 and 9, solutions of S702were very clear at pH 2 and 3, but were slightly hazy at pH 4.

Example 5

This Example contains an evaluation of the solubility in a soft drink(Sprite) and a sports drink (Orange Gatorade) of the soy protein isolateproduced by the method of Example I (S702). The solubility wasdetermined with the protein added to the beverages with no pH correctionand again with the pH of the protein fortified beverages adjusted to thelevel of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply I g of protein was weighed into abeaker and a small amount of beverage was added and stirred until asmooth paste formed. Additional beverage was added to bring the volumeto 50 ml, and then the solutions were stirred slowly on a magneticstirrer for 60 minutes to yield a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a LECO FP528 NitrogenDeterminator then an aliquot of each of the protein containing beverageswas centrifuged at 7800 g for 10 minutes and the protein content of thesupernatant measured in each sample.

Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) (3.39) and the sports drink (Orange Gatorade) (3.19)without protein was measured. A sufficient amount of protein powder tosupply 1 g of protein was weighed into a beaker and a small amount ofbeverage was added and stirred until a smooth paste formed. Additionalbeverage was added to bring the volume to approximately 45 ml, and thenthe solutions were stirred slowly on a magnetic stirrer for 60 minutes.The pH of the protein containing beverages was measured and thenadjusted to the original no-protein pH with HCl or NaOH as necessary.The total volume of each solution was then brought to 50 ml withadditional beverage, yielding a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a LECO FP528 NitrogenDeterminator then an aliquot of the protein containing beverages wascentrifuged at 7800 g for 10 minutes and the protein content of thesupernatant measured.

Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100

The results obtained are set forth in the following Table 9:

TABLE 9 Solubility of S702 in Sprite and Orange Gatorade no pHcorrection pH correction Solubility Solubility Solubility (%) Solubility(%) (%) in in Orange (%) in Orange Batch Product Sprite Gatorade inSprite Gatorade S005-L11-08A S702 100 100 96.4 100

As can be seen from the results of Table 9, the S702 protein wasextremely soluble in both the Sprite and the Orange Gatorade. Note thatthe S702 is a neutral pH product but the slightly higher pH of thenon-corrected beverage samples did not appear to negatively affect thesolubility.

Example 6

This Example contains an evaluation of the clarity in a soft drink andsports drink f the soy protein isolate produced by the method of Example1 (S702).

The clarity of the 2% w/v protein dispersions prepared in a soft drink(Sprite) and a sports drink (Orange (liatorade) in Example 5 wereassessed for clarity using the methods described in Example 4. For theabsorbance measurements at 600 nm, the spectrophotometer was blankedwith the appropriate beverage before the measurement was performed.

The results obtained are set forth in the following Tables 10 and 11:

TABLE 10 Clarity (A600) of S702 in Sprite and Orange Gatorade no pHcorrection pH correction A600 in A600 in A600 in Orange A600 in OrangeBatch Product Sprite Gatorade Sprite Gatorade S005-L11-08A S702 0.2090.520 0.158 0.204

TABLE 11 HunterLab haze readings for S702 in Sprite and Orange Gatoradeno pH correction pH correction haze haze (%) in haze haze (%) in (%) inOrange (%) in Orange Batch Product Sprite Gatorade Sprite Gatorade noprotein 0.0 44.0 0.0 44.0 S005-L11-08A S702 35.7 80.8 32.6 65.6

As can be seen from the results of Tables 10 and 11, despite theexcellent solubility, Sprite and Orange Gatorade samples containing S702were somewhat hazy. Correcting the pH reduced the haze level onlyslightly.

Example 7

This Example was conducted to extract the soy protein source withcalcium chloride solution at various pH values.

Three samples of defatted, minimally heat processed soy flour (10 geach) were extracted with 0.15M CaCl₂ (100 ml) for 30 minutes at roomtemperature with a magnetic stirrer/stir bar. One sample was extractedat natural pH, one sample was adjusted to pH 2.98 with dilute HCl andthe third sample was adjusted to pH 8.55 with dilute NaOH. The pH of theextraction systems was adjusted immediately after wetting the flour.After the extraction, the samples were centrifuged at 10,200 g for 10minutes to separate extract from the spent meal. The supernatant wasthen further clarified by filtration through a 0.45 μm pore size syringefilter. Filtrates were analyzed for pH, conductivity, clarity (A600) andprotein content (Leco). A sample of filtrate was also diluted 1:1 withan equal volume of RO water and the A600 measured again, Diluted andundiluted filtrate samples were acidified to pH 3 with diluted HCl andthe A600 measured again.

The properties of the filtrates obtained are set forth in the followingTable 12:

TABLE 12 Properties of initial extracts sample A600 % proteinExtractability (%) cond. (mS) natural pH 0.072 3.00 55.2 22.9 pH 2.980.109 3.88 71.5 27.9 pH 8.55 0.139 3.46 63.7 23.0

As may be seen in Table 12, the low pH conditions extracted the highestamount of protein. However, the extractability was quite good at all thepH conditions evaluated.

The clarity of the acidified, full strength extracts is set forth in thefollowing Table 13:

TABLE 13 Effect of acidification on clarity of full strength extractssample initial pH final pH final A600 natural pH 5.44 2.94 0.052 pH 2.983.10 3.10 0.109 pH 8.55 8.18 2.78 0.140

As can be seen from Table 13, upon acidification, all of the extractswere quite clear, but the sample extracted at natural pH was theclearest.

The clarity of the acidified, diluted extracts is set forth in thefollowing Table 14:

TABLE 14 Effect of acidification on clarity of diluted extracts sampleinitial pH initial A600 final pH final A600 natural pH 5.53 2.582 2.930.046 pH 2.98 3.22 0.056 2.81 0.050 pH 8.55 8.14 2.756 3.05 0.112

As may be seen from Table 14, when the samples were diluted 1:1 withwater and then acidified, all samples again were quite clear. However,the clarity of the samples extracted at natural and acidic pH was betterthan the sample extracted at high pH.

Example 8

This Example illustrates the production of soy protein isolate that issoluble, transparent and heat stable in acidic solutions and is membraneprocessed at natural pH then fractionated by a dilution step.

‘a’ kg of soy ‘b’ was added to ‘c’ L of 0.15 M CaCl₂ solution at ambienttemperature and agitated for 30 minutes to provide an aqueous proteinsolution. The residual soy protein source was removed and the resultingprotein solution was clarified by centrifugation and filtration toproduce L filtered protein solution having a protein content of ‘e’ % byweight.

‘f’ L of the protein extract solution was reduced to ‘g’ on a ‘h’membrane having a molecular weight cutoff of ‘i’ Daltons, producing aconcentrated protein solution with a protein content of ‘j’ % by weight.The concentrated protein solution was then diafiltered with ‘k’ L of0.15M CaCl₂ solution on the same membrane used for the initialconcentration step. The diafiltered protein solution was then furtherconcentrated to ‘l’ kg on the same membrane used for the initialconcentration and diafiltration steps, producing a concentrated proteinsolution with a protein content of wt %.

‘n’ kg of the concentrated or concentrated and diafiltered proteinsolution at ‘o’° C. was then diluted into reverse osmosis (RO) purifiedwater having a temperature of ‘q’° C. A white cloud formed immediatelyand was allowed to settle. The supernatant was removed by centrifugationand the precipitated protein was recovered in a yield of wt % of thefiltered protein solution. The recovered ‘s’ kg of protein precipitatewas then washed with about volumes of water and the water decanted. ‘u’of the washed precipitate was then resolubilized in about ‘v’ volumes ofwater with sufficient diluted hydrochloric acid added to adjust thesample pH to An additional ‘x’ kg of pH 3 RO water was added to thin theresolubilized precipitate to facilitate spray drying. kg of there-solubilized precipitate was then spray dried. The dried protein wasfound to have a protein content of ‘z’% (N×6.25) d.b. The product wasgiven the designation ‘aa’ S7300. Another ‘ab’ kg of the re-solubilizedprecipitate fraction was heated to 90° C. for 1 minute and then dilutedwith about ‘ac’ L of RO water to facilitate spray drying. The driedprotein was found to have a protein content of ‘ad’% (N×6.25) d.b. Theproduct was given a designation ‘aa’ S7300H. The other ‘ae’ of thewashed precipitate was resolubilized in about ‘af’ volumes of water withsufficient diluted phosphoric acid added to adjust the sample pH to‘ag’. ‘ah’ kg of the re-solubilized precipitate fraction was then spraydried. The dried protein was found to have a protein content of ‘ai’%(N×5.25) d.b. The product was given a designation ‘aa’ S7300-02. Theparameters ‘a’ to ‘ai’ are shown in the following Table 15.

TABLE 15 Parameters for runs to produce S7300 products S013/ aaS005-C19-09A S013-J06-09A S013-J27-09A 15-K30-09A a 20 50 40 40 b flour(defatted, white flake white flake white flake minimally heat processed)c 200 500 400 400 d 172.9 276.4 325 330 e 2.25 2.47 2.44 2.38 f 172.9275 325 330 g 19.7 kg 25.48 kg 22 kg 67 L h PES PES PES PES i 100,000100,000 100,000 100,000 j 16.36 22.06 not determined 9.74 k n/a n/a n/a335 l n/a n/a n/a 23.2 m n/a n/a n/a 23.7 n 19.7 25 22 22.7 o 31.5 2725.2 30 p 1:10 1:15 1:15 1:15 q 2.4 17.3 14.9 13 r 61.7 67.1 57.1 53.6 s5.01 8.26 9.78 10.7 t 0 2 2 2 u all all half all v 1 2 2 1.7 w 1.97 3.202.81 3 x 8.5 0 0 0 y 18.5 11.36 16.2 26 z 98.76 101.74 100.92 100.73 abn/a 12.57 n/a n/a ac n/a 26 n/a n/a ad n/a 101.60 n/a n/a ae n/a n/ahalf n/a af n/a n/a 2 n/a ag n/a n/a 2.76 n/a ah n/a n/a 12.4 n/a ai n/an/a 94.32 n/a n/a = not applicable

3.2% protein solutions of the S7300, S7300H and S7300-02 products wereprepared in water and the colour and clarity assessed using a HunterLabColorQuest XE operated in transmission mode. The pH of the solutions wasmeasured with a pH meter.

The pH, colour and clarity values are set forth in the following Table16.

TABLE 16 pH and HunterLab scores for 3.2% protein solutions of S7300,S7300H and S7300-02 batch sample pH L* a* b* haze (%) S005-C19-09A S73002.27 97.10 −1.88 11.04 0.0 S013-J06-09A S7300 3.01 95.08 −0.67 10.08 7.4S013-J06-09A S7300H 2.99 88.50 −0.29 9.00 42.4 S013-J27-09A S7300 2.7292.50 −0.60 10.17 29.6 S013-J27-09A S7300-G2 2.75 91.94 −0.23 9.51 36.3S013/15-K30-G9A S7300 2.92 95.90 −0.44 8.01 10.4

As may be seen by the results of Table 16, the pH of the S005-C19-09Aproduct ended up lower than the target pH of 3. This could be remediedby simply adding less acid when re-solubilizing the precipitate.Generally, these products produced lightly coloured solutions with highdegrees of transparency. The haze values obtained for the solution ofS013-J06-09A S7300H and the solutions of the S013-J27-09A products weresurprisingly high. It is thought that the haze present in these samplesmay have arisen from some difficulty in the spray drying process. Thefeed streams for these samples entering the spray dryer were quite clearas assessed by A600 measurement (data not shown). When the same 3.2% w/vprotein solutions of the 57300 products were evaluated on the HunterLabagain, one hour after preparation, the solutions were notably clearer asset forth in the following Table 17.

TABLE 17 pH and HunterLab scores for 3.2% protein solutions of S7300,S7300H and S7300-02 with measurement made one hour after solutionpreparation batch sample L* a* b* haze (%) S013-J06-09A S7300H 93.15−0.40 9.13 22.8 S013-J27-09A S7300 95.27 −0.80 9.62 10.0 S013-J27-09AS7300-02 94.63 −0.46 8.95 18.1

The colour of the dry powders was also assessed with the HunterLab inreflectance mode. The colour values are set forth in the following Table18.

TABLE 18 HunterLab scores for S7300, S7300H and S7300-02 dry powdersbatch sample L* a* b* S005-C19-09A S7300 86.43 −1.91 12.70 S013-J06-09AS7300 87.38 −1.09 10.61 S013-J06-09A S7300H 88.81 −0.82 8.00S013-J27-09A S7300 88.11 −1.04 11.97 S013-J27-09A S7300-02 88.09 −0.7311.31 S013/15-K30-09A S7300 88.17 −0.70 10.19

As may be seen from Table 18, the dry products were very light incolour.

The trypsin inhibitor activity of the S7300 products was determinedusing the method of Kakade et al, Cereal Chem., 51:376-381 (1974). Theresults obtained are shown in the following Table 19.

TABLE 19 Trypsin Inhibitor Activity (TIA) for S7300, S7300H and S7300-02in TIU/mg protein (N × 6.25) batch sample TIA S005-C19-09A S7300 49S013-J06-09A S7300 11.8 S013-J06-09A S7300H 3.1 S013-J27-09A S7300 37.7S013-J27-09A S7300-02 36.6 S013/15-K30-09A S7300 47.5

As may be seen from Table 19, the products prepared from the precipitateformed upon dilution of the concentrated protein solution had a lowertrypsin activity than was found in Example 1 for a product (S702)prepared similarly, but without the dilution step. The value of washingthe precipitate with water before re-solubilizing and drying is unclearbased on the variability in the results. A very low TIA was obtained byheat treating the re-solubilized precipitated protein. Comparing theresults in Table 19 to the trypsin inhibitor activity values for thesupernatants from the same dilution steps illustrates that the dilutiondoes fractionate the precipitated protein away from the trypsininhibitors. The trypsin inhibitor activities of the supernatants areshown in Table 20.

TABLE 20 Trypsin Inhibitor Activity (TIA) for unprocessed supernatantsin TIU/mg protein (N × 6.25) batch TIA S005-C19-09A not determinedS013-J06-09A 294.0 S013-J27-09A 219.2 S013/15-K30-09A 272.6

As may be seen from Table 20, the TIA of the supernatants was notablyhigher than the precipitate derived products.

Example 9

This Example illustrates methods of processing the supernatant streamsarising from the procedures of Example 8 to form additional soy proteinproducts.

The pH of the supernatant from the dilution step was adjusted from ‘a’to ‘b’ by the addition of diluted HCl. ‘c’ L of supernatant was thenreduced to ‘d’ kg on a ‘e’ membrane with a molecular weight cutoff of‘f’ Daltons. The concentrated protein solution had a proteinconcentration of ‘g’ wt %. With additional protein recovered from thesupernatant, the overall recovery of the filtered protein solution was‘h’%. kg of the concentrated supernatant was spray dried to form aproduct with a protein content of ‘j’ (N×6.25) d.b. The product wasgiven the designation ‘k’ S7200. ‘l’ kg of the concentrated supernatantwas adjusted to pH ‘m’ with diluted sodium hydroxide solution. kg of theconcentrated supernatant was then heat treated at 85° C. for 10 minutes,which precipitated about ‘o’% of the protein associated with theconcentrated supernatant. ‘p’ kg of precipitated protein was recoveredby centrifugation and washed with about ‘q’ volumes of RO water thenrecovered by centrifugation again. ‘r’ kg of washed precipitate wasfreeze dried to form a product with a protein content of ‘s’ % (N×6.25)d.b. This product was designated ‘k’ S7200P. The centrate containing theprotein not precipitated by the heat treatment was filtered and thenspray dried to form a product with a protein content of % (N×6.25) d.b.This product was designated ‘k’ S7200H. Parameters ‘a’ to T are setforth in the following Table 21.

TABLE 21 Parameters for the production of S7200 products from thedilution supernatants prepared as shown in Example 8 S013/ kS005-C19-09A S013-J06-09A S013-J27-09A 15-K30-09A a 6.26 5.66 5.74 5.82b 3.16 n/a 1.96 n/a c 200 370 355 335 d 5.34 19.96 20 19.74 e PES PESPES PES f 10,000 100,000 100,000 100,000 g 7.32 3.34 3.77 3.30 h 71.776.9 66.6 61.9 i 5.34 n/a n/a n/a j 91.66 n/a n/a n/a l n/a n/a 19.3 n/am n/a n/a 6.57 n/a n n/a 19.96 19.3 19.74 o n/a 61.2 71.6 69.2 p n/a2.42 3.08 2.58 q n/a 0 2 2 r n/a 2.06 2.70 2.14 s n/a 99.78 98.06 101.61t n/a 81.49 70.24 not determined n/a = not applicable

3.2% protein solutions of the S7200 and S7200H products were prepared inwater and the colour and clarity assessed using a HunterLab ColorQuestXE operated in transmission mode. The pH of the solutions was measuredwith a meter. The S7200P was poorly soluble and so the colour andclarity of this sample was not tested.

The pH, colour and clarity values are set forth in the following Table22.

TABLE 22 pH and HunterLab scores for 3.2% protein solutions of S7200 andS7200H batch sample pH L* a* b* haze (%) S005-C19-09A S7200 3.04 95.86−1.07 9.95 3.4 S013-J06-09A S7200H 5.80 95.82 −1.36 11.44 42.9S013-J27-09A S7200H 6.24 96.18 −0.93 9.82 23.9 S013/15- S7200H notK30-09A determined

As may be seen from Table 22, all the supernatant derived productsyielded lightly coloured solutions. However, the S013406-09A andS013-J27-09A products were hazier than the S005-C19-09A product. Thisdifference may be attributable to many different factors such asdifferences in pH, processing and soy protein source. However, the spraydrying issues mentioned in Example 8 may have played a role. Thecentrates arising from the removal of the heat deposited protein fromthe concentrated supernatant were filtered and quite clear as assessedby A600 measurement prior to the drying step.

The colour of the dry powders was also assessed with the Hunter ab inreflectance mode. The colour values are set forth in the following Table23.

TABLE 23 HunterLab scores for S7200 and S7200H dry powders batch sampleL* a* b* S005-C19-09A S7200 87.30 −0.21 8.13 S013-J06-09A S7200H 86.99−0.34 8.47 S013-J27-09A S7200H 85.97 −0.22 7.20 S013/15-K30-09A S7200Hnot determined

As may be seen from Table 23, the dry products were very light incolour.

The trypsin inhibitor activity of the supernatant derived products wasdetermined using the method of Kakade et al. Cereal Chem., 51:376-381(1974). The results obtained are shown in the following Table 24.

TABLE 24 Trypsin Inhibitor Activity (TIA) for S7200, S7200P and S7200Hin TIU/mg protein (N × 6.25) batch sample TIA S005-C19-09A S7200 482S013-J06-09A S7200P 78.6 S013-J27-09A S7200P 8.7 S013/15-K30-09A S7200P40.1 S013-J06-09A S7200H 296.7 S013-J27-09A S7200H 209.8 S013/15-K30-09AS7200H not determined

As may be seen from Table 24, the S7200P products had notably lowertrypsin inhibitor activities than the S7200H products. This suggeststhat the trypsin inhibitors remain soluble when the concentratedsupernatant is fractionated by heat induced precipitation. Lower TIAvalues for the S7200P were obtained when the protein precipitate waswashed with water before drying. The particularly low value obtained forthe S013-J127-09A S7200P may also be related to the pH regimen employedin that trial.

Example 10

This Example illustrates the production of soy protein isolate that issoluble, transparent and heat stable in acidic solutions that employsmembrane processing at natural pH and a dilution step, but the proteinfractions are not separated after dilution.

‘a’ ml of diafiltered and concentrated retentate from process runS013/S015-K30-09A, prepared as described in Example 8, at approximately‘b’° C. was diluted with ‘c’ml of RO water at approximately ‘d’° C. Awhite cloud formed but when the pH of the sample was lowered to ‘e’ withdiluted HCl the protein re-solubilized. The protein content of thediluted and acidified solution was ‘f’ wt %. The diluted and acidifiedprotein solution was reduced from a volume of ‘g’ ml to approximately‘h’ g on a ‘i’ membrane with a molecular weight cutoff of ‘j’ Daltons,providing a concentrated protein solution with a protein content of ‘k’wt %. After removing a small sample of the concentrated protein solutionfor analysis, ‘l’ g of the concentrated protein solution was freezedried to provide ‘m’ g of a product termed ‘n’ S7301-01, which had aprotein content of ‘o’ wt % w.b. The remaining ‘p’ ml of concentratedprotein solution was diafiltered with ‘q’ ml of RO water on the samemembrane as used for the concentration step. A total of ‘r’ g ofdiafiltered and concentrated protein solution was obtained, having aprotein content of ‘s’ wt %. ‘t’ g of this solution was freeze dried toyield ‘u’ g of a product termed ‘n’ S7301-02, which had a proteincontent of ‘v’% w.b. Parameters ‘a’ to ‘v’ are shown in the followingTable 25.

TABLE 25 Parameters for the production of S7301 products n trial 1 trial2 a 250 120 b 20 24 c 750 1320 d 22 22 e 3.16 3.06 f 6.35 2.27 g 9801422 h 492 257 i PES PES j 10,000 10,000 k 12.12 11.40 l 218.92 114.12 m26.86 12.03 o 91.27 99.69 p 250 120 q 1250 120 r 223.42 119.70 s 12.8511.17 t 198.80 105.02 u 25.83 10.78 v 95.19 100.40

3.2% protein solutions of the S7301 products were prepared in water andthe colour and clarity assessed using a HunterLab ColorQuest XE operatedin transmission mode.

The colour and clarity values are set forth in the following Table 26.

TABLE 26 HunterLab scores for 3.2% protein solutions of S7301-01 andS7301-02 batch sample L* a* b* haze (%) trial 1 S7301-01 93.67 −0.219.93 15.7 trial 1 S7301-02 94.13 −0.01 8.74 13.4 trial 2 S7301-01 94.76−0.19 8.41 15.9 trial 2 S7301-02 94.78 −0.13 8.36 15.5

As may be seen from Table 26, all the S7301 solutions had light colourand quite low haze values. The S7301-02 samples, which were diafiltered,were lighter, less green, less yellow and clearer than the S7301-01samples, which were not diafiltered. This effect of diafiltration wasmore pronounced in trial 1, where the initial dilution volume was lowerand more diafiltration volumes were employed. However, the samples oftrial 2, which had a larger dilution volume and only one volume ofdiafiltration were overall lighter, less yellow and higher in proteincontent.

Example 11

This Example contains an evaluation of the heat stability in water ofthe soy protein isolates produced by the methods of Example 8 (S7300)and Example 10 (S7301).

2% w/v protein solutions of S013/15-K30-09A S7300 and trial 1 S7301-02in water were produced and the pH adjusted to 3 with HCl. The clarity ofthe solutions was assessed by haze measurement with the HunterLabColorQuest XE instrument. The solutions were then heated to 95′C, heldat this temperature for 30 seconds and then immediately cooled to roomtemperature in an ice bath. The clarity of the heat treated solutionswas then measured again.

The clarity of the protein solutions before and after heating is setforth in the following Table 27:

TABLE 27 Effect of heat treatment on clarity of S7300 and S7301solutions haze (%) after product haze (%) before heating heatingS013/15-K30-09A S7300 6.4 4.2 trial 1 S7301-02 12.0 5.2

As can be seen from the data in Table 27, the samples were heat stable.The protein solutions were initially quite clear and the heat treatmentactually reduced the level of haze

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides analternative method based on extraction of soy protein from sourcematerial using aqueous calcium chloride solution, to obtain a soyprotein product which is soluble in acidic media and forms heat stable,transparent solutions therein. Modifications are possible within thescope of this invention.

What we claim is:
 1. A method of producing a soy protein product havinga soy protein content of at least about 60 wt % (N×6.25), dry weightbasis, which comprises: (a) extracting a soy protein source with anaqueous calcium salt solution to cause solubilization of soy proteinfrom the protein source and to form an aqueous soy protein solution, (b)separating the aqueous soy protein solution from residual soy proteinsource, (c) concentrating the aqueous soy protein solution whilemaintaining the ionic strength substantially constant by using aselective membrane technique, (d) optionally diafiltering theconcentrated soy protein solution, and (e) drying the concentrated andoptionally diafiltered soy protein solution.
 2. The method of claim 1wherein said calcium salt is calcium chloride.
 3. The method of claim 2wherein said calcium chloride solution has a concentration of less thanabout 1.0 M.
 4. The method of claim 3 wherein said calcium chloridesolution has a concentration of about 0.10 to about 0.15 M.
 5. Themethod of claim 1 wherein said extraction step is effected at atemperature of about 15° C. to about 35° C.
 6. The method of claim 1wherein said extraction step is carried out at a pH of about 5 to about11.
 7. The method of claim 6 wherein said pH is about 5 to about
 7. 8.The method of claim 1 wherein said aqueous soy protein solution has aprotein concentration of about 5 to about 50 g/L.
 9. The method of claim8 wherein said aqueous soy protein solution has a protein concentrationof about 10 to about 50 g/L.
 10. The method of claim 1 wherein saidaqueous calcium salt solution contains an antioxidant.
 11. The method ofclaim 1 wherein said aqueous soy protein solution is treated with anadsorbent to remove colour and/or odour compounds from the aqueous soyprotein solution,
 12. The method of claim 1 wherein said aqueous soyprotein solution is concentrated to a protein concentration of about 50to about 400 g/L.
 13. The method of claim 12 wherein said aqueous soyprotein solution is concentrated to a protein concentration of about 100to about 250 g/L,
 14. The method of claim 1 wherein said concentrationstep is effected by ultrafiltration using a membrane having a molecularweight cut-off of about 3,000 to about 1,000,000 Daltons.
 15. The methodof claim 14 wherein said concentration step is effected byultrafiltration using a membrane having a molecular weight cut-off ofabout 5,000 to about 100,000 Daltons.
 16. The method of claim 1 whereinsaid diafiltration step is effected using an aqueous calcium saltsolution of about the same pH and about equal or lower molarity than theextraction salt solution on the soy protein solution before or aftercomplete concentration thereof.
 17. The method of claim 16 wherein saiddiafiltration step is effected using about 2 to about 40 volumes ofdiafiltration solution.
 18. The method of claim 17 wherein saiddiafiltration step is effected using about 5 to about 25 volumes ofdiafiltration solution.
 19. The method of claim 16 wherein saiddiafiltration is effected using a membrane having a molecular weightcut-off of about 3,000 to about 1,000,000 Daltons.
 20. The method ofclaim 19 wherein said membrane has a molecular weight cut-off of about5,000 to about 100,000 Daltons.
 21. The method of claim 16 wherein saiddiafiltration is effected until no significant further quantities ofcontaminants or visible colour are present in the permeate.
 22. Themethod of claim 16 wherein said diafiltration is effected until theretentate has been sufficiently purified so as, when dried, to provide asoy protein isolate with a protein content of at least about 90 wt %(N×625) d.b.
 23. The method of claim 16 where an antioxidant is presentduring at least part of the diafiltration step.
 24. The method of claim1 wherein said concentration step and optional diafiltration step arecarried out at a temperature of about 2° to about 60° C.
 25. The methodof claim 24 wherein said temperature is about 20° to about 35° C. 26.The method of claim 1 wherein the concentration and optionaldiafiltration step are operated in a manner favourable to the removal oftrypsin inhibitors.
 27. The method of claim 1 wherein the concentratedand optionally diafiltered soy protein solution is treated with anadsorbent to remove colour and/or odour compounds.
 28. The method ofclaim 1 wherein the concentrated and optionally diafiltered soy proteinsolution is subjected to a pasteurization step prior to drying.
 29. Themethod of claim 28 wherein said pasteurization step is effected at atemperature of about 55° to about 70° C. for about 30 seconds to about60 minutes.
 30. The method of claim 29 wherein said pasteurization stepis effected at a temperature of about 60° to about 65° C. for about 10to about 15 minutes.
 31. The method of claim 28 wherein saidpasteurized, concentrated and optionally diafiltered soy proteinsolution is cooled to a temperature of about 15° C. to about 35° C. fordrying or further processing.
 32. The method of claim 1 wherein theconcentrated and optionally diafiltered soy protein solution isacidified to a pH of about 2.0 to about 4.0 prior to drying.
 33. Themethod of claim 32 wherein said acidified soy protein solution issubjected to a heat treatment step to inactivate heat-labileanti-nutritional factors prior to drying.
 34. The method of claim 33wherein the anti-nutritional factors are heat-labile trypsin inhibitors.35. The method of claim 33 wherein the heat treatment step alsopasteurizes the aqueous protein solution.
 36. The method of claim 33wherein said heat-treatment is effected at a temperature of about 70° toabout 120° C. for about 10 seconds to about sixty minutes.
 37. Themethod of claim 36 wherein said heat-treatment is effected at atemperature of about 85° to about 95° C. for about 30 seconds to about 5minutes.
 38. The method of claim 33 wherein the heat-treated clearacidified soy protein solution is cooled to a temperature of about 2° toabout 60° C. for drying or further processing.
 39. The method of claim38 wherein the heat-treated clear acidified soy protein solution iscooled to a temperature of about 20° to about 35° C. for drying orfurther processing.
 40. The method of claim 1 wherein a reducing agentis present during the extraction step to disrupt or rearrange thedisulfide bonds of trypsin inhibitors to achieve a reduction in trypsininhibitor activity.
 41. The method of claim 1 wherein a reducing agentis present during the concentration and/or optional diafiltration stepto disrupt or rearrange the disulfide bonds of trypsin inhibitors toachieve a reduction in trypsin inhibitor activity.
 42. The method ofclaim 1 wherein a reducing agent is added to the concentrated andoptionally diafiltered soy protein solution prior to drying and/or thedried soy protein product to disrupt or rearrange the disulfide bonds oftrypsin inhibitors to achieve a reduction in trypsin inhibitor activity.43. The method of claim 1 wherein said soy protein product has a proteincontent of about 60 to about 90 wt % (N×6.25). d.b.
 44. The method ofclaim 1 wherein said soy protein product has a protein content of atleast about 90 wt % (N×6.25). d.b.
 45. The method of claim 1 whereinsaid soy protein product has a protein content of at least about 100 wt% (N×6.25). d.b.
 46. A soy protein product produced by the method ofclaim
 1. 47. An acidic solution having dissolved therein the soy proteinproduct of claim
 46. 48. The aqueous solution of claim 47 which is abeverage.
 49. The soy protein product of claim 46 which is blended withwater-soluble powdered materials for the production of aqueous solutionsof the blend.
 50. The blend of claim 49 which is a powdered beverage.